Metanephric kidney glomerular capillary walls develop from tbe formation of microvasculature, close apposition of immature endothelial and visceral epithelial cells (podocytes), and fusion of a dual basement membrane between these two cell layers. With subsequent capillary loop expansion, additional basement membrane, derived mainly from podocytes, is spliced into the fused GBM. Although many of the morphological features of glomerular development have been described, very little is known about the exact origins of the microvasculature and how basement membrane assembly occurs. The three specific aims in this renewal application will begin to define the cellular and molecular mechanisms operative in glomerulogenesis in vivo. (1) The origins of the glomerular endothelium and mesangium will be determined. This will be accomplished by grafting fetal mouse kidneys from normal/transgenic donors into transgenic/normal host mice. In situ hybridization will define the glomerular cell lineages within these grafts. (2) Binding sites within the developing GBM for certain laminin domains are hypothesized to be important for basement membrane fusion and splicing. This idea will be tested by domain-specific mAb affinity isolation of proteolytic laminin fragments, injection of these into newborn rodents, and immunoelectron microscopy. In addition, a laminin cleaving enzyme identified in developing glomeruli will be characterized and its role in GBM assembly determined. (3) Using antibodies against recombinant laminin cloned from a fetal rat kidney expression library, the cellular origin, distribution, and fate of authentic kidney laminin within the developing glomerulus will be defined. These experiments will provide fundamentally new information on mechanisms of glomerular capillary growth and GBM assembly.